Insulin Represses Transcription of the Thyroid Stimulating Hormone β-Subunit Gene through Increased Recruitment of Nuclear Factor I

Abstract

Although the regulation of thyroid stimulating hormone β-subunit gene (TSHβ) has been intensively studied, the functions of transcription factors involved are not fully understood. The authors found that the −615/−516 promoter region of the TSHβ interacts specifically with nuclear proteins derived from pituitary tissue or from cultured thyrotroph cells. The actual binding site at the nucleotide level, as revealed by DNase I protection assay, includes the consensus sequence for nuclear factor I (NFI). RT-PCR analysis indicated that NFI-B expression is restricted to thyrotroph cells in the anterior pituitary. EMSA and ChIP analysis showed that NFI-B binds most efficiently to the −588/−560 region of TSHβ promoter. The forced expressions of NFI-B markedly reduced TSHβ promoter activity and its mRNA expression. Furthermore, it was also shown that the −588/−560 region is involved in the insulin-mediated repression of the TSHβ. It was of particular interest to observe that NFI-B was recruited to the −588/−560 region of the TSHβ promoter in an insulin-dependent manner. Taken together, this study provides new insights of the delicate regulations of energy metabolism and hormonal homeostasis.

Introduction

To maintain homeostasis, endocrine cells must be capable of responding to changing hormonal environments. In general, cellular responsiveness is accomplished via a signal transduction cascade that ultimately alters gene expression. In the hypothalamus-pituitary-thyroid axis, thyrotropin releasing hormone (TRH)⁵ from the hypothalamus stimulates the expression of TSH in the anterior pituitary, which in turn stimulates the release of the thyroid hormones (THs), T3 and T4. THs function to regulate metabolism, growth, development, and reproduction, and excessive level of THs regulate the production of TSH via feedback inhibition. Thus, the expression of TSH is essentially regulated by TRH and THs in thyrotroph cells of the anterior pituitary (1–3).

TSH consists of glycoprotein hormone α-subunit (αGSU) and β-subunit, but TSH levels are mainly dependent on the regulated expression of the TSHβ (4). Many previous studies on the hormone-regulated expression of the TSHβ have focused on its regulation by TRH and THs. However, there is a growing recognition that other hormone-induced changes in TSHβ expression are important. Furthermore, it was recently reported that insulin and insulin-like growth factor (IGF) are expressed in the pituitary, which raises the possibility that some glycoprotein hormones are regulated in an insulin-dependent manner in the anterior pituitary (5, 6). At the cellular level, THs stimulate mitochondrial oxygen consumption and increase thermogenesis and influence glucose metabolism (7–9). Therefore, the TSHβ is a strong candidate for insulin-targeted gene in the anterior pituitary. Indeed, there are evidences that insulin and IGF affect the hypothalamus-pituitary-thyroid axis, but insulin has not been directly linked to TSH (10, 11).

The transcription factor NFI has been implicated in hormonal and signal transduction pathways involving insulin, TGF-β, cAMP, steroid hormones, vitamin-D, vitamin-B6, TNF-α, TSH, FSH, DNA-PK, and others (12). Insulin stimulates glucose uptake by many cells via a complex cascade of signaling events, and it affects GLUT4 gene transcription via NFI in adipose cells (13–15). Furthermore, NFI has been shown to mediate the repression of the 5-aminolevulinate synthase gene by insulin (16). However, no evidence has been presented regarding the direct influence of insulin on the binding of NFI protein to target gene promoters. In mammals, the NFI family consists of four subtypes (NFI-A, -B, -C, and -X), which are expressed in complex, overlapping patterns during embryogenesis (12). Subtypes of NFI are thought to be involved in the regulation of developmental and tissue-specific gene expression. These subtypes are DNA-binding proteins that interact specifically with the dyad-symmetric binding sites (TTGGCN₅GCCAA) of the promoters of target genes. Dimerization of NFI to homo- or hetero-dimer is essential for its DNA binding and transcriptional activities (17). In the hypothalamus and anterior pituitary, NFI has been shown to be involved in the transcriptional regulations of GnRH and Lhx3 (18, 19). Therefore, based on NFI expression and activity in the anterior pituitary, it seems likely that NFI plays a role in the regulation of anterior pituitary-related genes.

In the present study we identified a novel element that contributes to the thyrotroph-specific expression of the TSHβ. Furthermore, we demonstrate that this element is responsible for the down-regulation of the TSHβ by NFI and insulin. These results provide a new mechanism for the insulin-mediated regulation of TSHβ in a thyrotroph-specific manner.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection

TαT1, LβT2, and αT1-1 cells, which were derived from thyrotroph, gonadotroph, and progenitor of thyrotroph cells in mouse anterior pituitary, respectively, were generous gifts from Dr. Pamela Mellon (University of California, San Diego). These cells were grown in DMEM supplemented with 10% fetal bovine serum. TαT1 and αT1-1 cells were seeded on Matrigel-coated plates (BD Biosciences) to facilitate adhesion (20). Lacto-somatotropic GH₃ cells were grown in monolayer culture in DMEM containing 2.5% heat-inactivated fetal bovine serum and 15% heat-inactivated horse serum. 293T cells were grown in monolayer culture in DMEM containing 10% heat-inactivated fetal bovine serum. All cells were grown at 37 °C in 5% CO₂ incubator in accordance with the routine cell culture procedures. Cultured cells were transfected by the polyethyleneimine technique as previously described (21). Luciferase activities were measured using an LB 953 Autolumat (EG&G Berthold) as previously described (22, 23). β-Galactosidase assay was performed colorimetrically by a standard protocol (24). Luciferase activities were normalized based on the expression of Rous sarcoma virus-β-galactosidase plasmid.

Plasmids, siRNA, and Reagents

The initial TSHβ-luciferase reporter construct contains −1099 to +13 bp of rat TSHβ promoter linked to the luciferase gene in the plasmid pLuc-link2 (25). Two 5′-deletion constructs (−615 and −516) were prepared using SpeI and HincII restriction enzyme sites, which are uniquely present in TSHβ promoter region. An internal deletion construct lacking NFI binding site was prepared as follows. KpnI to EcoRI fragment spanning −795 to −580 region was PCR-amplified using forward primer (5′-CCAGGTACCTTAGATAAACAGTGATC-3′) and reverse primer (5′-CCCGAATTCAACTGACCTGAGATCAAAT-3′). EcoRI to NheI fragment spanning −568 to +13 region was also PCR-amplified using forward primer (5′-CCCGAATTCGATTTAGCCACGCTATCAG-3′) and reverse primer (5′-CATGCCTGCAGGTCGACTGGCTAGCCAGG-3′). These two PCR products were simultaneously ligated into the pLuc-link2 vector, which was cut with KpnI and NheI restriction enzymes. The mutant clone resulting from a three-fragment ligation was confirmed by DNA sequencing analysis. Multimers of −588/−560 region of TSHβ promoter were subcloned upstream of a Rous sarcoma virus promoter that was linked to the luciferase coding sequence (25). The pCHNFA1.1, pCHNFIB2, pCHNFIC2, and pCHNFIX2 plasmids expressing hemagglutinin (HA)-tagged mouse NFI-A, -B, -C, and -X, respectively, were kindly provided by Dr. R. M. Gronostajski (University of New York, Buffalo, NY). Depletion of NFI-B was performed using specific siRNA duplex from Santa Cruz Biotechnology targeted to NFI-B. Control siRNA-A, a non-targeting 20–25-nucleotide siRNA from Santa Cruz Biotechnology, served as a negative control. Insulin, Genistein, and other chemicals were obtained from Sigma.

Sequence Alignment and Transcription Factor Cluster Analysis

TSHβ promoter sequences of the human, chimpanzee, mouse, rat, and dog were aligned by mVISTA software. Transcription factor cluster analysis was processed using Transfac databases.

Quantitative Real Time RT-PCR

A two-step real-time PCR was carried out to analyze the mRNA expression of candidate genes. Total RNA was reverse-transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. The reaction was primed by 12–15 oligo(dT) primer. The oligonucleotide primers used in the quantitative real time RT-PCR were as follows. TSHβ forward (5′-TCTGTGCTGGGTATTGTATGAC-3′) and reverse (5′-GCGGCTTGGTGCAGTAGTTG-3′) primers were designed to amplify the 249-bp fragment. αGSU forward (5′-GCAGCTGTCATTCTGGTCATG-3′) and reverse (5′-CTACGACTTGTGGTAGTAGCA-3′) primers were designed to amplify the 353-bp fragment. Primers to detect NFI subtypes were prepared as described previously (19). β-Actin mRNA, which served as an internal control, was amplified with β-actin forward (5′-ATCGTGGGCCGCCCTAGGCA-3′) and reverse (5′-TGGCCTTACCCTTCAGAGGGG-3′) primers. Real-time PCR was performed using the Rotergene Real-Time analysis system (Rotor-Gene 2000 Robocycler, Corbett Research) according to the manufacturer's protocol with SYBR Green as a fluorescent dye. All reactions were performed under identical conditions using 35 cycles of amplification with denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s, and elongation at 72 °C for 40 s. The specificity of products generated by each set of primers was examined using gel electrophoresis and further confirmed by a melting curve analysis. The relative expression level was computed with respect to the expression level of the internal standard, β-actin mRNA.

Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts were prepared from rat pituitary tissue, TαT1 cells, GH₃ cells, and NFI-B-transfected 293T cells essentially as described (26). DNA fragments from TSHβ promoter region were end-labeled with [α-³²P]dCTP by DNA polymerase I filling-in reaction. 25 μl of reaction mixture contained 20,000 cpm of DNA probe, 4 μg of nuclear extracts, 1 μg of BSA, and various amount of poly(dI-dC) in 25 mm Tris-HCl (pH 8.0), 5 mm MgCl₂, 50 mm KCl, 1 mm EDTA, 1 mm DTT, 5% glycerol. The reaction mixtures were incubated for 30 min at room temperature, electrophoresed through a nondenaturing polyacrylamide gel in 0.5× Tris borate EDTA at 4 °C, and then analyzed by autoradiography. For supershift assays, 0.8 μg of anti-HA antibody was added to the reaction mixture before electrophoresis.

DNase I Protection Assay

DNA-protein binding reactions were done as described above and then subjected to the DNase I digestion as follows. 30 min after the binding reaction, 0.075 units of RQ1 DNase I (Promega) in 100 mm Tris-HCl (pH 7.5), 35 mm MgCl₂ was added and incubated for an additional 5 min. The reaction was terminated by the addition of 3 μl of 0.5 m EDTA, extracted with phenol, and then ethanol-precipitated. The precipitated products were electrophoresed through an 8 m urea denaturing gel and analyzed by autoradiography.

Chromatin Immunoprecipitation (ChIP) Assay

ChIP assay was performed using the reagents from Upstate Biotechnology Inc. according to the manufacturer's protocol with the following modifications. About 5 × 10⁶ TαT1 cells that were transfected with pCHNFI-A, -B, -C, -X and NFI-B siRNA were treated with 1% formaldehyde for 10 min at room temperature and then quenched with 0.125 m glycine. After washing with PBS, cells were lysed with 0.4 ml of SDS lysis buffer. The lysate was sonicated 15 times for 10 s each using a 150 W Sonifier Cell Disrupter 185 (Branson) at a 10% power setting. The size of chromatin fragments was checked on a 2% agarose gel. This sonication method consistently yielded 200–2000-bp fragments with the majority being ∼500 bp long. For each immunoprecipitation, 0.1 ml of the sonicated cell lysate was diluted with 0.1 ml of SDS lysis buffer. The 0.2-ml diluted lysate was then further diluted 10-fold in ChIP dilution buffer. The lysate was immunoprecipitated with anti-HA antibody (Roche Diagnostic) and anti-NFI antibody (Santa Cruz Biotechnology). Immune complex was collected with protein A-agarose (Sigma). Immunoprecipitated DNA and input DNA were subjected to PCR using a primer set that amplified −700 to −450 region of TSHβ promoter which includes putative NFI binding site (forward, 5′-TCTAGGAACAGGAACACATAC-3′; reverse, 5′-TAATAAAAACATTTTAGTTCA-3′).

Preparation of Cell Extracts and Immunoblot Analysis

Whole cell extracts were prepared using radioimmune precipitation assay buffer (Sigma) supplemented with a protease inhibitor mixture (Sigma). For immunoblot analysis, the protein samples were transferred to a nitrocellulose membrane following SDS-PAGE. The primary antibodies used in this study were mouse monoclonal anti-HA antibody (Roche Diagnostic) and mouse monoclonal anti-tubulin antibody (Sigma). Binding of antibodies was detected by the SuperSignal system (Pierce).

RESULTS

The −615/−516 Region of TSHβ Promoter Specifically Interacts with Nuclear Extracts Derived from Thyrotroph Cells in Vitro

To examine the interactions of transcription factors that might mediate the thyrotroph-specific expression of TSHβ, EMSA was employed using DNA fragments from the −1099 to +176-bp region of TSHβ promoter. Each probe was prepared using restriction enzymes, EcoRI, KpnI, HincII, BstNI, TaqI, and PstI, which cleave at −1099, −795, −516, −172, −79, and, +176 sites of the gene, respectively. Although several protein-DNA complexes can be detected by a long exposure of the film, the −795/−516 fragment clearly appeared to bind to nuclear extracts in a thyrotroph-specific manner (Fig. 1A). To narrow down the binding region, the −795/−516 fragment was further digested with SpeI, which cleaves at the −615 site. EMSA again demonstrated the binding of the −615/−516 fragment with thyrotroph nuclear extracts (Fig. 1B). In the presence of excess unlabeled −615/−516 fragment, the DNA-protein complex band completely disappeared (Fig. 1C). These results indicate that thyrotroph-specific protein physically interacted with the −615/−516 region of TSHβ promoter. To identify the DNA binding sequence involved, the −615/−516 DNA fragment was subjected to a DNase I footprinting assay (Fig. 2). The footprints produced using both sense and antisense strands showed a region that was protected by the TαT1 nuclear extracts. Interestingly, this region contains a 5′-TGGCATCCTGCCA-3′ sequence, a typical NFI DNA binding sequence.

FIGURE 1.

Thyrotroph-specific protein bound to the −615/−516 region of TSHβ promoter in vitro. A, five restriction fragments were isolated from the TSHβ promoter region and examined for nuclear protein binding by EMSA. 10 μg of nuclear extracts from rat pituitary cells, TαT1cells, and GH₃ cells were incubated with each labeled DNA probe in the presence of 0.5 or 1 μg of poly(dI-dC). B, two DNA fragments from the −795/−516 region of TSHβ promoter were also examined for the binding of TαT1 nuclear extracts (N.E.) by EMSA. C, competition assays (Comp.) were performed by adding a 100–500-fold molar excess of unlabeled −615/−516 fragment.

FIGURE 2.

Thyrotroph-specific protein bound to the −590/−563 region of TSHβ promoter. DNase I protection assays were performed using the −615/−516 region of TSHβ promoter. After incubating radiolabeled DNA with 10 μg of TαT1 nuclear extracts, reaction mixtures were partially digested with DNase I and then analyzed by denaturing PAGE and autoradiography. The DNA fragments used in the binding reaction were also subjected to the Maxam and Gilbert chemical sequencing reactions to produce a size standard. A, protected sequences are marked with lines in the sense (S, +) and antisense (AS, −) strands. B and C, boxes on the right side of each panel indicate region protected by TαT1 nuclear extracts (N.E.).

The 5′-TGGCATCCTGCCA-3′ Sequence Is Highly Conserved in Mammalian TSHβ Promoters

The progressive releases of genomic sequences make it possible to use phylogenetic footprinting to compare promoter regions among species. To identify the putative promoter sequences of the TSHβ, mVISTA software was used for multiple alignment analysis versus several known mammalian TSHβ promoter sequences, including those of human, chimpanzee, mouse, rat, and dog. Although the most conserved sequences are located in an approximately −100-bp region of TSHβ promoter, a second region of homology is also apparent in the −591/−563 region of rat TSHβ promoter (dotted box in Fig. 3A and shaded in Fig. 3B). This region also contains the sequence of the DNA binding site of the thyrotroph-specific protein identified in Fig. 2. Transcription factor cluster analysis revealed that NFI site is located in the −580/−568 region. These findings suggest that NFI is involved in the regulation of TSHβ expression in thyrotroph cells.

FIGURE 3.

A bioinformatic approach revealed conserved regions in mammalian TSHβ promoter. The 1000-bp promoter sequences of mammalian TSHβs of human, chimpanzee, mouse, rat, and dog were analyzed using mVISTA software. The sequences shown were obtained from the UCSC genome browser. A, pairwise alignments are shown in which conserved regions with a high score are drawn as peaks. B, mammalian TSHβ promoter sequences were compared in a multiple alignment format. The conserved region, which contains a binding site (underlined) for the transcription factor NFI, is shaded for all the species.

NFI-B Expression Is Restricted to Thyrotroph Cells

The NFI gene family consists of four genes, NFI-A, -B, -C, and -X. Because sequence data located the binding of NFI to TSHβ promoter, RT-PCR was performed to examine the expression patterns of NFI-A, -B, -C, and -X in various pituitary-derived cell lines, namely, GH₃ (a somato-lactotroph), LβT2 (a gonadotroph), αT1-1 (a thyrotroph precursor), and TαT1 (a mature thyrotroph) cells. Fig. 4 clearly shows that NFI-B was expressed only in thyrotrophic TαT1 cells. These expression patterns agree well with those reported by two other groups (18, 27).

FIGURE 4.

The expressional patterns of NFI subtypes were anterior pituitary cell type-dependent. RT-PCR was performed using total RNA from TαT1, GH₃, LβT2, and αT1-1 cells, and the products obtained were analyzed on agarose gel. β-Actin was used as an internal control.

NFI-B Specifically Interacts with the −588/−560 Region of TSHβ Promoter

To determine whether NFI can bind to TSHβ promoter in vivo, we performed ChIP assays on TSHβ promoter using an NFI antibody. Result showed that NFI binds to the promoter region of TSHβ, whereas control IgG did not precipitate detectable DNA (Fig. 5A). Furthermore, to examine whether NFI subtypes have different degrees of the binding activity for TSHβ promoter, chromatin was individually isolated from TαT1 cells transfected with four different NFI subtypes. ChIP assays demonstrated that NFI-B specifically binds to the TSHβ promoter region, which contains the putative NFI binding site (Fig. 5B). NFI-C also binds to this region but to a lesser degree. We focused on the NFI-B for further study as it strongly binds to the TSHβ promoter. To establish the physical interaction between NFI-B and the −588/−560 region of TSHβ promoter, the −588/−560 oligonucleotide was synthesized and used in EMSA (Fig. 5C). It was observed that nuclear extracts from 293T cells transfected with HA-tagged NFI-B expression plasmid produced three-slowly migrating complexes (Fig. 5C, second through fourth lanes). The addition of an excess unlabeled oligonucleotide greatly reduced these complexes (Fig. 5C, fifth lane) and indicated that the binding was specific. In the presence of anti-HA antibody, only the slowest-migrating complex, which was most prominent, was supershifted (Fig. 5C, sixth lane). Again, the supershifted band completely disappeared after the addition of an excess of unlabeled oligonucleotide (Fig. 5C, seventh lane). Because complex-1 and 2 were not supershifted by anti-HA antibody, it may suggest that endogenous NFI proteins or other factors in 293T cells are involved in these complexes (18). Next, siRNA was employed against NFI-B to further verify the specific binding of endogenous NFI-B. After the NFI-B-specific siRNA (siNFI-B) transfection, NFI-B expression decreased specifically at the mRNA level when compared with the control siRNA transfection (Fig. 5, D and E). Upon depletion of NFI-B using siRNA, the immunoprecipitation signal decreased significantly, indicating that endogenous NFI-B indeed binds to this promoter region of the TSHβ (Fig. 5F).

FIGURE 5.

NFI-B bound to the −588/−560 region of TSHβ promoter in vitro and in vivo. A, antibody (Ab) against NFI was used in a ChIP assay followed by PCR to amplify −700 to −450 region of TSHβ promoter from genomic DNA isolated from TαT1 cells. Mouse genomic DNA (Input) served as a positive control for PCR. Normal IgG was included in the assay as a negative control. B, TαT1 cells transfected with expression plasmids for each HA-tagged NFI subtype were examined in a ChIP assay where anti-HA antibody was used. C, EMSA was performed using a radiolabeled oligonucleotide containing the −588/−560 sequence. 1, 5, and 10 μg of nuclear extracts (N.E.) containing HA-tagged NFI-B were incubated with a probe and separated on a non-denaturing gel. Competition assays were done by adding a 500-fold molar excess of unlabeled oligonucleotide to the binding reaction. A supershift assay was also carried out, where anti-HA antibody was added to the binding reaction. S.S. indicates a super-shifted complex. ND, not determined, D, TαT1 cells were plated onto a 6-well plate, and control (Con.) or NFI-B (siNFI-B) siRNAs were transfected 36 h before total RNA extraction. RT-PCR was performed to detect NFI subtypes and β-actin mRNA. E, real-time PCR was performed to measure the NFI mRNA levels from cDNA samples obtained from D. NFI mRNA levels were normalized by β-actin mRNA expression levels. F, a ChIP assay was performed as in A with cells transfected with control siRNA or NFI-B-specific siRNA for 36 h.

NFI-B Represses TSHβ Expression in Thyrotrophic TαT1 Cells

To determine how each of the NFI subtypes is involved in TSHβ expression, TSHβ mRNA level was examined using RT-PCR and quantitative real-time PCR after the forced expression of each NFI subtype. Surprisingly, overexpression of NFI-B or NFI-C resulted in 80 and 70% decreases in TSHβ mRNA, respectively, suggesting that they might act as repressors of TSHβ (Fig. 6). To define the region of TSHβ promoter necessary for these repressions by NFI subtypes, a series of 5′-deletion constructs was transfected into TαT1 cells together with an expression plasmid of each NFI subtype. The promoter activity of the −615 construct, which contains the putative NFI binding site, was found to be repressed by NFI-B or NFI-C overexpression but not by the overexpression of the other NFI subtypes (Fig. 7A). Furthermore, no such repression was observed for the −516 construct, which does not contain the putative NFI binding site. The importance of the putative NFI binding site was confirmed when a construct in which an internal deletion was introduced at positions −580/−568 was compared with a wild type construct (Fig. 7B).

FIGURE 6.

NFI repressed TSHβ expression. A and B, TαT1 cells were transfected with 1 μg of expression plasmid for each NFI subtype for 24 h. Total RNA was then isolated, and TSHβ mRNA expression levels were determined by RT-PCR and quantitative real-time PCR using β-actin as an internal control. C, immunoblot analysis with anti-HA antibody confirmed forced NFI expression in transfected TαT1 cells. Tubulin was used as a protein loading control.

FIGURE 7.

The −580/−568 region of TSHβ promoter was involved in the NFI-mediated repression of the TSHβ. A, TαT1 cells were transiently transfected with 1 μg of a −615 or −516 TSHβ construct together with 0.5 μg of expression plasmid for each NFI subtype. B, 1 μg of the wild type or internal deletion mutant of the TSHβ construct was transfected into TαT1 cells together with 0.5 μg of expression plasmid for each NFI subtype. C, 1 μg of multimer construct was transfected into TαT1 and GH₃ cells together with 0.5 μg of expression plasmid for NFI-B or NFI-C. TSS, transcription start site. D, 1 μg of the −615 or −516 constructs was transfected into TαT1cells together with Lhx2, NFI-B, and NFI-C expression plasmids as indicated. Each transfection was performed in triplicate, and data are expressed as the -fold expressions relative to the controls ± S.E.

To prove that the putative NFI binding site is sufficient to confer NFI-B- or NFI-C-mediated repression, multiple copies of the −588/−560 sequence were subcloned upstream of the Rous sarcoma virus promoter fused to luciferase reporter gene and transfected into TαT1 and GH₃ cells. In TαT1 cells, basal Rous sarcoma virus promoter activity decreased as the number of copies of the −588/−560 region increased until no more repression was observed upon overexpressing NFI-B or NFI-C (Fig. 7C, left). Interestingly, promoter activity in GH₃ cells, which do not express NFI-B, was similarly inhibited by exogenous NFI-B but not by NFI-C (Fig. 7C, right). These results support the notion that NFI-B is absolutely required for the repression of the TSHβ and that the −588/−560 region is sufficient for its repression in TαT1 cells.

Because the transcription factor Lhx2 has been reported to act as an activator of TSHβ (28), cotransfection experiments were carried out in which the −615 TSHβ construct or the −516 TSHβ construct was transiently transfected into TαT1 cells together with combinations of Lhx2 and NFI-B or NFI-C expression plasmids (Fig. 7D). Lhx2 activated TSHβ promoter as mentioned above, and this was then repressed by the overexpression of NFI-B or NFI-C in −615 construct but not in the −516 construct. Thus, both Lhx2 and NFI may provide mechanisms that finely regulate the expression of the TSHβ.

NFI-B Is Involved in the Repression of TSHβ by Insulin in Thyrotroph Cells

Because NFI has been reported to mediate the repression of several genes by insulin (15, 16), quantitative real-time PCR was employed to determine the affects of insulin on TSHβ expression in thyrotroph cells via NFI. TαT1 cells were cultured in the indicated concentrations of insulin, and the mRNA expression levels of the αGSU and TSHβ were determined. Fig. 8A shows that TSHβ mRNA levels were decreased dose-dependently by insulin, and αGSU mRNA levels were slightly decreased. Transfection experiments demonstrated that −615 construct activity was inhibited by insulin, whereas the activity of the −516 construct and TK (thymidine kinase) promoter activity were unaffected (Fig. 8B). Next, we examined the involvement of NFI-B in the regulation of TSHβ by insulin. The results clearly demonstrated that depletion of NFI-B increased the TSHβ mRNA levels and abolished the repressing activity of insulin (Fig. 8, C and D). To delineate the mechanisms responsible for modulating the expression of the TSHβ by insulin, the expression patterns of NFI subtypes were examined in the presence or absence of insulin. However, the mRNA expression levels of NFI subtypes were not altered by insulin treatment in the 1–1000 nm dose range.⁶ ChIP assays were used to investigate changes in NFI-B recruitment by TSHβ promoter in vivo. Notably, insulin treatment increased NFI-B binding to TSHβ promoter in non-targeting siRNA-treated control but not in NFI-B-depleted cells (Fig. 9A). These results demonstrate that insulin exerts its effects by modulating the binding activity of NFI-B on the TSHβ promoter in thyrotroph cells. Furthermore, the cells transfected with a construct having an internal deletion of NFI binding site did not respond to either NFI-B or insulin treatment (Fig. 9B). This observation raised the possibility that NFI-B is strongly involved in the insulin-mediated repression of the TSHβ.

FIGURE 8.

Insulin repressed transcription of the TSHβ via NFI-B. A, TαT1 cells were incubated at the indicated insulin concentrations for 12 h. Total RNA was isolated, and expressions of TSHβ and αGSU mRNA were measured by quantitative real-time PCR. β-Actin mRNA was used as an internal control. B, TαT1 cells were transiently transfected with 1 μg of the −615 or −516 TSHβ constructs for 24 h. Cells were treated with 100 nm of insulin for 12 h. At least three individual experiments were performed in triplicate. Values are -fold inductions and are presented as the means ± S.E. Thymidine kinase (TK) promoter fused to a luciferase gene was served as a negative control. C, TαT1 cells transfected with control (Con.) or NFI-B (siNFI-B) siRNAs were treated with vehicle or 100 nm insulin for 12 h before total RNA extraction. RT-PCR was performed to detect TSHβ and β-actin. D, real-time PCR was performed to measure the TSHβ mRNA levels from cDNA samples obtained from C. TSHβ mRNA levels were normalized by β-actin mRNA expression levels.

FIGURE 9.

Insulin increased the NFI-B binding to the TSHβ promoter. A, TαT1 cells transfected with control (Con.) or NFI-B (siNFI-B) siRNAs were treated with vehicle or 100 nm insulin for 12 h before immunoprecipitation. A ChIP assay was performed using antibody against NFI-B. Mouse genomic DNA Input served as a positive control for PCR. Normal IgG was included in the assay as a negative control. Ab, antibody. B, 1 μg of the wild type or an internal deletion mutant of TSHβ promoter construct was transfected into TαT1cells with 0.5 μg of NFI-B expression plasmid for 12 h. TαT1 cells were then treated with 100 nm of insulin and/or 1 μm of genistein (Genis.) for another 12 h. Each set of transfections was performed in triplicate, and values are expressed as -fold repressions (mean ± S.E.) versus basal activity for three independent experiments.

Because NFI has been reported to be phosphorylated in adipocytes treated with insulin (15), we also examined the effects of genistein, an inhibitor of tyrosine protein kinase, on TSHβ promoter activity in the presence of NFI-B or insulin. Genistein not only stimulated basal TSHβ promoter activity but also restored TSHβ promoter activity that was previously repressed by NFI-B or insulin (Fig. 9B). On the other hand, genistein treatment did not result in any significant changes in the activity of internal deletion mutant of TSHβ construct. Because this inhibitor may work at the downstream of insulin signaling cascades, we also examined the expressions of insulin receptor, insulin receptor substrate (IRS) 1, 2, 3, and 4 and insulin receptor-related receptor in TαT1 cells in response to genistein treatment. Expression levels of these factors did not significantly change.⁶ However, IRS1 mRNA level slightly increased by genistein treatment in TαT1 cells, which agreed with a previous report in MCF-7 cells (29). Taken together, these results suggest that the phosphorylation of NFI protein may be a key process during the insulin-mediated repression of TSHβ in thyrotroph cells.

DISCUSSION

In the present study, which was undertaken to identify regulators of the TSHβ, we initially found that nuclear extracts of TαT1 thyrotroph cells interact with the TSHβ promoter in an in vitro binding assay. Transcriptional regulation of the TSHβ plays a fundamental role in determining TSH expression levels in a thyrotroph-specific manner. In this regard, a TαT1 cell line that was derived from a thyrotroph of mouse anterior pituitary seemed to be an excellent model system for a cell type-specific gene regulation (20). We considered that the identification of thyrotroph specific cis-acting elements and trans-acting factors would provide useful information concerning the mechanism of TSHβ regulation and an insight of the delicate mechanism underlying the regulation of TSH. Interactions between transcription factors and specific sequence motifs in the promoter region of genes are fundamental to such processes. These motifs can be bound by cell type-specific transcription factors with varying activities, which enables transcriptional regulation to be specifically modulated by different cellular signals. In the present study we identified a novel element that can be bound by the transcription factor NFI and might contribute to the thyrotroph-specific expression of the TSHβ. It has been found that NFI can alter the activities of anterior pituitary related genes, such as GH, GnRH, and Lhx3 (18, 19, 27, 30, 31). In addition, the transcription factor Pitx2, which plays an essential role in pituitary development, is regulated by NFI expression (32). Although all NFI subtypes are expressed in anterior pituitary tissue, transcripts of NFI-B are not detected in sommato-lactotroph cells; likewise, transcripts of NFI-A and NFI-B are not detected in gonadotroph cells at the cellular level (18, 19, 27). Interestingly, NFI-A and NFI-B were differentially expressed from αT1-1 cells (progenitor cells of thyrotroph, sommato-lactotroph, and gonadotroph) to mature thyrotroph (TαT1) and gonadotroph cells (LβT2) (Fig. 4). These results suggest that differential expressions of NFI subtypes (A, B, C, and X) are developmentally regulated in the anterior pituitary. The four different isoforms of NFI may have different effects on transcription of the target genes in an adult anterior pituitary. NFI-B is ubiquitously expressed in most tissues, including the pituitary. However, NFI-B knock-out mice showed severe defects in lung maturation and brain development, resulting in lethality (33–35). Moreover, it is unknown whether levels of thyroid hormone-related hormones and insulin are changed in this mouse. Further study will be required to elucidate the exact function of NFI-B for hormone regulation in pituitary using the mouse model system.

According to our EMSA and ChIP assay results, NFI-B strongly binds to TSHβ promoter. Furthermore, it was interesting to see that although NFI-C bound to TSHβ promoter in ChIP assays, it was not observed in EMSA.⁶ In addition, although NFI-B and NFI-C were observed to repress TSHβ promoter activities in TαT1 cells, NFI-C could not repress TSHβ promoter activity in GH₃ cells, which do not express endogenous NFI-B (Fig. 7C). Taken together, these results support the notion that TSHβ repression by NFI-C depends on the presence of NFI-B in thyrotroph cells. This may be due to the indirect or weak interaction between NFI-C and TSHβ promoter. More study is required to determine how NFI isoforms selectively influence TSHβ regulation and to identify the mechanisms responsible for the repression of TSHβ expression by specific NFI subtypes. Several possible mechanisms were suggested for the activation and repression activities of NFI (12). It is likely that repression and activation by NFI are both cell type-specific and context-specific of target promoter. As shown in Fig. 7, we did not observe any significant increase of promoter activity in multimer constructs by increasing the copy number of the −580/−560 region, which contained the NFI binding site. Thus, our findings did not support direct competition between NFI and other trans-acting factors. In our previous study, we reported that Lhx2 can activate TSHβ expression in thyrotroph cells (28). Lhx2 activity was remarkably repressed by the overexpression of NFI-B or NFI-C (Fig. 7D). This result suggested that NFI-B could repress the TSHβ gene expression by contacting different components of the basal transcription complex. It will be of interest to determine whether repression by NFI-B is mediated through changes in chromatin structure or whether it depends on the contexts of transcription factors at the TSHβ promoter (36). Indeed, TSHβ is regulated at the level of chromatin structure (37).

The synthesis and secretion of TSH are controlled by external stimuli, such as those provided by TRH, T3, or T4 (1–3). Because of the physiological importance of THs, many studies have been conducted on its regulation of the TSHβ. However, reports on the negative regulation of the TSHβ have been restricted to TH-dependent feedback regulation (3, 37–41). Recently, it was discovered that insulin and IGF are expressed in the pituitary (5, 6). Insulin is known to play a central role in glucose-related homeostasis and in the expression of over 100 genes (42–44). The physiological significance of the insulin role in hypothalamus-pituitary-thyroid has not been established in detail, but certain clues suggest its importance. THs activities strongly affect the plasma insulin concentration (45, 46). Insulin also modulates thyroid hormone activity in some aspects (47, 48). Interestingly, it was reported that TSH response to TRH injection failed in Type II diabetes patients (49). It seems likely that insulin and THs share the functions for some aspect of glucose metabolism, and they can regulate each other's expression. These reports suggest that insulin may influence the TSHβ expression in anterior pituitary, thus regulating THs.

Although some evidence suggests that insulin can modulate the expression of TSH, the molecular mechanism involved has not been established (50). In the present study we found that insulin modulates the expression of TSHβ via NFI and that the TSHβ is down-regulated by insulin in a dose-dependent manner (Fig. 8, A and B). This repression of the TSHβ by insulin was absent in a clone with a deletion of NFI binding site in TSHβ promoter (Fig. 8D). These results support the hypothesis that the NFI binding site is required for the insulin to affect TSHβ expression. On the other hand, insulin treatment did not affect the mRNA expression levels of NFI subtypes in TαT1 cells.⁶ Therefore, it is likely that post-translational modification of NFI-B protein or some another factor is required for the repression of TSHβ by insulin. Thus far, it has not been reported previously that the phosphorylation statuses of NFI proteins can directly control their DNA binding activities. Of particular interest is our finding that insulin increased binding of NFI-B protein to TSHβ promoter (Fig. 8C). We also found that genistein rescued TSHβ repression by NFI and by insulin. These results support one possible mechanism, namely, that insulin can influence the DNA binding activity of NFI-B protein by changing the phosphorylation status of NFI-B.

Based on these results, we hypothesize that excess insulin levels down-regulate TSH synthesis in the anterior pituitary followed by the down-regulation of THs in the thyroid. It can be considered that the regulation between insulin and TSHβ is central to the negative feedback mechanism.